Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States with an estimated prevalence of 30 to 40% of the adult population. Although only 5 to 20% of NAFLD patients are generally considered to meet histopathologic criteria for nonalcoholic steatohepatitis (NASH), this still translates into a nationwide prevalence of 2 to 5% of the population who are at an increased risk of progression to cirrhosis. Cirrhosis itself is a sufficient risk factor for liver cancer including hepatocellular carcinoma (HCC). Obesity also appears to contribute to the risk of developing HCC with a meta-analysis of cohort studies showing a 90% increased risk of HCC in the obese. This may partially explain the growing rates of HCC in developed countries and the 80% increase in the annual incidence of HCC in the U.S. over the last two decades (Torres et al., Semin Liver Dis., 2012, 32(1):30-38).
Even more alarming is the fact that HCC has been shown to occur in patients having noncirrhotic NASH. Cumulatively, this data provides strong evidence that HCC can occur more in noncirrhotic NASH patients than in cirrhotic NASH patients, although larger prospective studies are required to obtain incidence rates. The connection between NASH and liver cancer has been well established. Large case studies have demonstrated a specific phenotype of older, predominantly male patients with coexisting metabolic syndrome that can develop HCC without a background of cirrhosis. Some of the purported complex pathophysiologic mechanisms that lead to this have been described, but a total understanding of the interplay and overlap between NASH promoting and oncogenic processes is still in development (Torres et al., Semin Liver Dis., 2012, 32(1):30-38).
Major causes of hepatitis and HCC can be divided into the following two categories: (1) infections with hepatitis B (HBV) and C viruses (HCV), and (2) metabolic causes, such as alcohol consumption and NAFLD. Generally, chronic viral infection-mediated hepatitis is the most common cause of hepatitis, followed by alcoholic liver diseases and NAFLD. Drug therapy of viral infection-mediated hepatitis is already established and the anti-viral therapy using interferon alpha and nucleotide analogs is commonly prescribed. On the other hand, drug therapy of NAFLD, NASH and diseases associated with liver cancer have not been established since the disease itself was only recently recognized.
Obesity has become a worldwide health problem and is known to increase the risk of diabetes, cardiovascular diseases, and several types of cancer. Among obesity-associated cancers, liver cancer has been shown to have a strong relationship with obesity, based on epidemiological studies (Bhaskaran et al., Lancet, 2014, 384: 755-765; Calle and Kaaks, Nature Reviews Cancer, 2004, 4:579-591; and Calle et al., New England J. Med., 2003, 348(17):1625-1638). The most common risk factor for HCC is long-term infection by HBV or HCV (El-Serag, New England J. Med., 2011, 365:1118-1127; and Marengo et al., Annual Review of Medicine, 2016, 67:103-117). However, obesity-associated NAFLD and NASH have recently emerged as risk factors for liver cancer (Marengo et al., Annual Review of Medicine, 2016, 67:103-117; Michelotti et al., Nat. Rev. Gastroenterol. Hepatol., 2013, 10:656-665; and Streba et al., World J. Gastroenterology, 2015, 21(14):4103-4110). There is no available therapeutic for NAFLD, NASH and NASH-associated liver cancer at present. Therefore, there is an urgent need for the development of a therapeutic for NASH-associated liver cancer.
Prostaglandins are mediators of pain, fever and other symptoms associated with inflammation. Prostaglandin E2 (PGE2) is the predominant eicosanoid expressed in inflammation conditions. PGE2 is also involved in various physiological and/or pathological conditions, such as hyperalgesia, uterine contraction, digestive peristalsis, awakeness, suppression of gastric acid secretion, blood pressure, platelet function, bone metabolism, angiogenesis, and cancer cell growth, invasion and metastasis, or the like. Non-patent references disclose the character of the prostanoid receptors, relationship with therapy, and selective agonists and antagonists most generally used (see, for example, Konya et al., Pharmacology & Therapeutics, 2013, 138:485-502; and Yokoyama et al., Pharmacol. Rev., 2013, 65:1010-1052).
PGE2 has been reported to be highly expressed in cancer tissues of various types of cancer, and it has also been demonstrated that PGE2 correlates to the initiation, growth and development of cancer and disease conditions of patients. It is generally accepted that PGE2 relates to activation of cell proliferation and cell death (apoptosis) and plays an important role in the processes of cancer cell proliferation, disease progression and cancer metastasis (see, for example, Konya et al., Pharmacology & Therapeutics, 2013, 138:485-502; and Yokoyama et al., Pharmacol. Rev., 2013, 65:1010-1052).
There are four PGE2 receptor subtypes, EP1, EP2, EP3 and EP4, which display different pharmacological properties. The EP4 receptor subtype belongs to the G protein-coupled receptor subfamily, known as a receptor with seven transmemblene domains. Accordingly, EP4 plays a significant role in biological events by stimulating cAMP signal-mediated functions. From the aspect of pharmacological studies, several investigations of compounds with EP4 receptor antagonistic activities have been conducted and EP4 receptor-selective antagonists are known (Konya et al., Pharmacology & Therapeutics, 2013, 138:485-502).
Regarding the role of the EP4 receptor in cancer, several non-patent references (e.g., Yokoyama et al., Pharmacol. Rev., 2013, 65:1010-1052; Ma et al., Oncolmmunology, 2013, 2(1):e22647) and patent literature (U.S. Pat. No. 8,921,391 B2) demonstrate the growth inhibitions and/or metastasis of the colon, breast, gastric, lung, prostate, and other cancer types in animal tumor models using EP4 receptor antagonists. Some patent literature (e.g., WO 2015/179615 A1 and US 2015/0004175 A1) shows therapeutic efficacy of an EP4 receptor antagonist or inhibition of EP4 signaling results in the inhibition of tumor growth. Moreover, EP4 signal inhibition in combination with other anti-cancer therapeutics or radiation therapy show additional benefits compared to each monotherapy (see WO 2015/179615 A1).
The role of the EP4 receptor in liver cancer has recently been reported in non-patent literature. PGE2/EP4 receptor signaling through PKA/CREB activation upregulated c-Myc expression and resulted in promoting cell growth in HCC cells in vitro (Xia et al., Oncology Reports, 2014, 32:1521-1530). PGE2 also promoted a hepatic stellate cell-induced myeloid-derived suppressor cell (MDSC) accumulation in in vitro and in vivo experiments which was supposed to stimulate growth of liver cancer (Xu et al., Oncotarget, 2016, 7(8):8866-8878). This literature indicated that PGE2/EP4 signaling may have some role in liver cancer growth. However, these references do not demonstrate liver cancer suppression of PGE2/EP4 signal inhibition in animals. Suppression of PGE2/EP4 receptor signaling alone (or in combination with a PD-1 antibody) restores CD8+ T cell (CTL) functional activity (Chen et al., Nature Medicine, 2015, 21(4):327-334; and US 2015/0004175 A1). These references demonstrate that EP4 signal inhibition mediates activation of a host's CTL activity, but there is no direct evidence that EP4 signal inhibition or EP4 antagonistic activity has efficacy on HCC growth and/or metastasis.
U.S. Pat. No. 8,921,391 B2 demonstrates anti-tumor efficacies of Compound A, B, and/or C in gastric, lung, prostate, and other cancer types in animal tumor models. In this patent, liver cancer is noted as one of the cancer types in a one-word description without any experimental example, and liver cancer does not appear in any claim. Additionally, this patent does not disclose the treatment of “nonalcoholic steatohepatitis (NASH)-associated” liver cancer, and does not disclose any information relating to NASH or NAFLD.
In 2015, a critical concern of PGE2/EP4 signal inhibition in liver cancer therapy, especially in chronic virus-infection related diseases, such as HBV and HCV-mediated liver diseases, was reported. Inhibiting PGE2/EP4 signal causes significant induction or activation of PD-1 expression in virus-specific CD8+ T cells (CTLs) in an animal model (Chen et al., Nature Medicine, 2015, 21(4):327-334). Increase of PD-1 expression on CTLs strongly suggests a suppression of key immunological function mediated by T cells against viral infection. In the case of chronically HBV- or HCV-infected liver cancers, the increase of PD-1 expression on CTLs should therefore cause a stimulation of viral expansion and also tumor development and growth. The impact of the increase of PD-1 expression on CTLs in tumor growth and development has been clearly demonstrated by the striking efficacy of their inhibitor (e.g., an anti-PD-1 antibody or an immune checkpoint inhibitor) in recent clinical cancer therapy. Accordingly, this research created a general concern for the risk of increasing tumor growth by PGE2/EP4 signal inhibition in the treatment of liver cancer. Thus, PGE2/EP4 signal inhibition was expected to activate PD-1 expression, which would suppress the immunological response against a viral infection, and then promote virus development and liver cancer development.
In view of this negative concern for EP4 signal inhibition in liver cancer therapy, the present inventors have unexpectedly found significant anti-tumor effects and suppression of PD-1 expression on CD8+ T cells (CTLs) in NASH-associated liver cancer. This is contrary to the results on PD-1 expression in a virus-associated liver cancer model for an EP4 antagonist in monotherapy and in combination with another drug in an animal model. NASH-associated liver cancer has different causes from virus infection-associated liver cancer. To date, there is no evidence to support the EP4 mechanism, including EP4 antagonistic activity, in a therapy to treat NASH-associated liver cancer. Moreover, no evidence has been disclosed in the art relating to the efficacy of a combination therapy of the EP4 receptor with any other therapies in the treatment of NASH-associated liver cancer. Accordingly, the use of an EP4 antagonist on NASH-associated liver cancer is unexpected over the art.